Part:BBa_M36222:Design
ArsAction: Arsenite Removal Device (input: PoPs Signal, output: removal device)
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 319
Illegal EcoRI site found at 709
Illegal PstI site found at 1487 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 319
Illegal EcoRI site found at 709
Illegal PstI site found at 1487 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 319
Illegal EcoRI site found at 709 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 319
Illegal EcoRI site found at 709
Illegal PstI site found at 1487 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 319
Illegal EcoRI site found at 709
Illegal PstI site found at 1487
Illegal NgoMIV site found at 1873
Illegal AgeI site found at 472 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
In designing this part, we had to take into consideration the form of arsenic we wanted to remove. Removing arsenic from a cell called for the insertion of another coding sequence that coded for the gene, arsC. This addition would have significantly increased the total base pair count of our device. Instead, we decided to have our device remove arsenite. Furthermore, the ars family of genes is usually regulated by the arsR operon, but we did not include a coding sequence for arsR in our design. Because we designed our actuator to respond to the PoPS signal, we decided that inclusion of the arsR operon as a regulatory operon would be redundant. Therefore, we removed it.
Also, it was necessary to codon optimize each part of our device in order to ensure successful transformation and use in the K-12 strain of E. Coli.
No promoter is necessary in our construct, because of the nature of this actuator. ArsAction responds to the polymerase per second (PoPS) signal so it is activated upon receiving this signal.
Ribosome binding sites were placed upstream of each gene coding sequence. The stronger RBS is upstream of the arsB sequence to ensure transcription of arsB does not taper off towards the end of the transcription of the actuator. The device needs both arsA and arsB in order to work so varying the strength of the binding sites ensures this.
Source
Genecopia. ArsA gene coding sequence. http://www.genecopoeia.com/product/search/view_features.php?cid=&prod_id=S0530
Biocyc. ArsB sequence. http://biocyc.org/ECOLI/sequence?object=EG12236
Information about Ars family of genes. Chenl, Chih-Ming, T K Misrat, S Silver, and B Rosen (1986). Nucleotide Sequence of the Structural Genes for an Anion Pump. Journal of Biological Chemistry. Vol 26 no. 31 pp.15030-15038.